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Biomedical subjects

R Cramer

Publications and source records attributed to R Cramer.

At least 19 recordsLinked to original sources
PubMed1992

A simple and rapid method for isolation of eosinophilic granulocytes from human blood.

A simple and rapid method for the purification of morphologically and functionally intact eosinophils from human blood of both normal and eosinophilic subjects is described. The method is based on a single centrifugation of total blood leukocytes suspended in Percoll with specific gravity 1.0853 g/ml, after erythrocyte removal by dextran sedimentation. The peculiarity of this isolation technique is the maintenance of strictly physiological values of pH and osmolality throughout the entire procedure. Moreover, the cells are not subjected to measures aimed at changing the physical properties of either neutrophils or eosinophils. Because of such characteristics, this isolation method could be usefully exploited for comparative studies of normal and eosinophilic normodense eosinophils and of neutrophils and eosinophils from the same noneosinophilic subject.

Cell Separation↗
PubMed1992

A new, one-step assay on whole cell suspensions for peroxidase secretion by human neutrophils and eosinophils.

The degranulation of neutrophils and eosinophils is frequently monitored by assaying myeloperoxidase (MPO) and eosinophil peroxidase (EPO) activity in the cell-free supernatant of degranulating cells, after removal of the cells by centrifugation. This procedure leads to underestimation of the extent of degranulation, since both peroxidases tend to stick to cell surfaces, to test tube walls, and to particulate stimuli used to elicit degranulation, because of their highly cationic nature. In this paper we describe a method for assaying MPO and EPO secretion in whole cell suspensions that avoids separation of the cells from the incubation medium. The least toxic and thus safest among the sensitive peroxidase substrates, 3,3',5,5'-tetramethylbenzidine (TMB), was employed for peroxidase assay. The method we describe here is applied to the detection of peroxidase release by neutrophil and eosinophil cell suspensions incubated in either polypropylene test tubes or flat-bottomed microtiter plate wells. Because of the omission of the centrifugation step, the TMB method offers two major advantages over the currently used techniques: (1) higher estimates of degranulation, which permits the use of a smaller number of cells (in the microassay version, 150,000 neutrophils and 50,000 eosinophils) and smaller amounts of the secretagogues, and (2) rapidity, since the degranulation assay can be performed immediately on completion of the cell incubation with the secretagogue.

Calcimycin↗
PubMed1992

Matrix-assisted laser desorption/ionization mass spectrometry of nucleic acids with wavelengths in the ultraviolet and infrared.

A number of different matrices have been tested and compared for ultraviolet and infrared (UV and IR) matrix-assisted laser desorption/ionization (MALDI) of oligodeoxyribonucleotides and mixtures thereof, as well as ribonucleic acids (tRNA from yeast and rRNA from E. coli). A new technique for removing alkali cations from nucleic acid samples during sample preparation on the sample support is demonstrated. The amount of oligonucleotide sample consumed during a typical measurement in IR-MALDI-MS was determined.

Angiotensin II↗
PubMed1991

Eosinophil activation on biologic surfaces. Production of O2- in response to physiologic soluble stimuli is differentially modulated by extracellular matrix components and endothelial cells.

Production of O2- in response to FMLP, TNF, IFN-gamma, platelet activating factor, LPS, substance P, and PMA by human eosinophils in suspension and in contact with polystyrene ELISA plastic (PL) or biologic surfaces was studied. Monolayers of human endothelial cells (HEC) or PL coated with FCS, fibronectin, laminin, collagen types I and IV, fibrinogen, or fibrin were used as biologic surfaces. Only PMA and FMLP stimulated O2- generation by eosinophils in suspension. Eosinophils residing on HEC monolayers, either untreated or treated with LPS, were unresponsive to all stimuli except PMA. PMA induced O2- generation by eosinophils on all surfaces; FMLP on all surfaces but HEC monolayers; TNF and platelet-activating factor only on PL, fibrinogen, and fibrin; LPS and substance P only on PL. PMA was equally effective on eosinophils on surfaces and in suspension, whereas the effect of FMLP was greater on eosinophils on surfaces than on eosinophils in suspension. IFN-gamma was ineffective on any of the surfaces tested. These results indicate that biologic surfaces may profoundly affect the ability of eosinophils to respond with a respiratory burst to physiologically relevant soluble stimuli, the effect varying according to the nature of both the stimulus and the surface. Since the respiratory burst generates products of oxygen reduction that are toxic to several tissue components, it follows that biologic surfaces may modulate eosinophil-induced tissue injury.

Endothelium, Vascular↗
PubMed1991

Oxidation of homovanillic acid as a selective assay for eosinophil peroxidase in eosinophil peroxidase-myeloperoxidase mixtures and its use in the detection of human eosinophil peroxidase deficiency.

Biochemical assays for peroxidase activity do not usually distinguish between different peroxidases. The guaiacol assay, for example, which is one of the most commonly used assays for peroxidase activity, is sensitive to both eosinophil peroxidase (EPO) and the peroxidase of neutrophils, i.e., myeloperoxidase (MPO), thus preventing distinction of the two peroxidases in mixed neutrophil-eosinophil populations. In this paper we describe a simple and sensitive method for selective assays of EPO in EPO-MPO mixtures or mixed populations of neutrophils and eosinophils. The method is based on the peroxidase-mediated oxidation of homovanillic acid (HVA) under appropriate assay conditions in which EPO is still very active in catalyzing the reaction whilst MPO-mediated HVA oxidation is almost undetectable. Optimal assay conditions were as follows: pH 10.5, 10 microM hydrogen peroxide, 0.8 mM HVA and an incubation time of 120 min at 37 degrees C. Under these conditions the assay permits EPO activities as low as 0.025 guaiacol U/ml to be measured even in the presence of 0.175 guaiacol U/ml of MPO. In mixed neutrophil-eosinophil cell suspensions the test permits the detection of as few as 5 X 10(3) eosinophils even in the presence of about 700 X 10(3) neutrophils (eosinophils: neutrophils ratio 1:140) with no appreciable interference by the latter cells. The method described here has been applied to studies of human EPO deficiency and proved to be successful in the identification of individuals with partial EPO deficiency, which is not feasible with non quantitative methods (for example, cytochemistry) or unselective biochemical assay of peroxidase activity.

Eosinophil Peroxidase↗
PubMed1991

Mechanisms of eosinophil adherence to cultured vascular endothelial cells. Eosinophils bind to the cytokine-induced ligand vascular cell adhesion molecule-1 via the very late activation antigen-4 integrin receptor.

We have examined the mechanisms involved in the adherence of normal peripheral blood eosinophils to cultured human umbilical vein endothelial cells (HEC) under three conditions: (a) adherence in the absence of treatment of HEC or eosinophils with activating agents (basal adherence); (b) adherence induced by stimulation of eosinophils with phorbol ester (eosinophil-dependent adherence); and (c) adherence induced by pretreatment of HEC with LPS, tumor necrosis factor (TNF), or IL-1 (endothelial-dependent adherence). A mechanism was identified that was equally active in basal, eosinophil-dependent, and endothelial-dependent adherence. This mechanism was optimally active in the presence of both Ca++ and Mg++, and reduced in the presence of Ca++ only or Mg++ only. Furthermore, like the other mechanisms of eosinophil adherence, it was active at 37 degrees C but not at 4 degrees C. A second mechanism of adherence was involved in eosinophil- and in endothelial-dependent adherence. This mechanism was dependent on the CD11/CD18 adhesion complex of eosinophils (i.e., inhibited by anti-CD18 MAb) and it was active in the presence of Ca++ and Mg++ or Mg++ only, but not Ca++ only. The third mechanism of adherence was specific for endothelial-dependent adherence. It involved the endothelial ligand vascular cell adhesion molecule-1 (VCAM-1) and the eosinophil receptor very late activation antigen-4 (VLA-4, CD49d/CD29, i.e., inhibited by anti-VCAM-1 MAb or anti-VLA-4 MAb). This mechanism was active in the presence of Ca++ and Mg++ but not of Ca++ only or Mg++ only, and was not up- or downregulated when eosinophils were stimulated with phorbol ester. In contrast, the endothelial leukocyte adhesion molecule-1 (ELAM-1), that binds neutrophils and monocytes, was not involved in eosinophil adherence to LPS-, TNF-, or IL-1-stimulated HEC (i.e., not inhibited by anti-ELAM-1 MAb). We conclude that eosinophils, like monocytes and lymphocytes, bind to the cytokine-induced endothelial ligand VCAM-1 via the integrin receptor VLA-4.

Antibodies, Monoclonal↗
PubMed1991

Effect of biological surfaces on neutrophil O2- production and its relationship to the CD11b/CD18 integrin-dependent adherence.

The production of O2- in response to LPS, PAF, FMLP, TNF and PMA by human neutrophils in suspension and residing on surfaces coated with fetal calf serum (FCS), fibronectin (FN), laminin (LM), collagen types I and IV (CI and CIV), fibrinogen (FBG) or fibrin (FBN) was studied. Of the agonists used, PAF and LPS failed to induce a response in any of the above conditions; FMLP and PMA stimulated neutrophils to produce similar amounts of O2- either in suspension or on biological surfaces; TNF induced O2- production only by cells residing on FN, FBG and FBN. These results indicate that production of oxygen-derived free radicals by neutrophils depends on the type of agonist and the nature of the surface they interact with. The relationship between the respiratory burst and adherence was studied by measuring O2- release and adherence of neutrophils residing on FN, LM, CIV, and FBG, in the absence and in the presence of the monoclonal antibody 60.3 that recognizes the common beta-chain of CD11/CD18 integrins. FMLP, PMA and TNF increased neutrophil adherence on all these surfaces except CIV. The monoclonal antibody markedly inhibited the FMLP and PMA-induced adherence but had no effect on the O2- release elicited by these two agonists. In contrast, the monoclonal antibody inhibited both the increased adherence and O2- release induced by TNF on FN and FBG. The TNF-induced increase in adherence to LM, that was not accompanied by an increase in O2- release, was also inhibited by the monoclonal antibody. We conclude that the respiratory burst of neutrophils residing on surfaces is not necessarily correlated with adherence.

Antigens, CD↗
PubMed1990

Mutual influence between eosinophil peroxidase (EPO) and neutrophils: neutrophils reversibly inhibit EPO enzymatic activity and EPO increases neutrophil adhesiveness.

The effects of the interactions between eosinophil peroxidase (EPO) and neutrophils were studied. It is shown that binding of EPO to neutrophils results on the one hand in reversible inhibition of EPO peroxidase activity and, on the other, in an increased neutrophil aggregation and neutrophil adhesion to endothelial cell monolayers. After prolonged periods of exposure to EPO, neutrophils also show a decreased ability to exclude the vital dye erythrosin B. The reversible inhibition of EPO activity may represent a means of keeping under control the oxidative potential of EPO, while the increased neutrophil aggregation and adherence to endothelial cells suggest that EPO may influence at least two key events of inflammation, that is, the margination of neutrophils and/or their accumulation in the inflammatory site.

Cell Adhesion↗
PubMed1989

Dual effect of a protein-free hemodialysate on the oxygen uptake of phagocytosing human polymorphonuclear leucocytes.

The effect of a protein-free hemodialysate (Solcoseryl) on the oxygen uptake of human polymorphonuclear leucocytes (PMN) was studied. No effect was observed on resting PMN over a concentration range from 0.5 to 10% v/v. Instead, when PMN phagocytosing serum opsonized zymosan (SOZ) were used, low concentrations (0.5-2.5% v/v) of the hemodialysate increased their oxygen uptake, whereas higher concentrations (5-10% v/v) were inhibitory. At a concentration of 10% the hemodialysate was shown to inhibit also SOZ particles uptake. These findings suggest that inhibition of the oxygen uptake might be related to inhibition of phagocytosis. The mechanism of the stimulatory effect observed at low concentrations remains to be established but it does not seem to be related to a direct activation of the biochemical pathway/s linked to O2 consumption since the hemodialysate per se had no effect on the respiration of resting PMN. The results are discussed in the light of the known clinical effects of the hemodialysate.

Actihaemyl↗
PubMed1986

An improved method to quantitate phagocytosis with mineral oil particles which avoid cell flotation.

We have shown that actively phagocytosing human polymorphonuclear leukocytes (PMN) float on top of the incubation medium when Oil Red O containing paraffin oil particles (density 0.8740) are used as the phagocytosable material. This implies that the quantitation of phagocytosis based on the recovery of Oil Red O in phagocytosing cells pelleted after centrifugation would be underestimated. We therefore prepared particles of progressively increasing density by mixing paraffin oil (density 0.8740) with silicon oil (density 1.0802). Cell flotation also occurred with paraffin oil-silicon oil particles and could be avoided only when the density of the particles used had reached 0.9952 g/cm3. Paraffin oil-silicon oil particles of a density sufficient to dissolve the dye Oil Red O were therefore used to quantitate both the initial rate of phagocytosis and the phagocytic capacity of human PMN. With this assay the initial rate of phagocytosis was found to be 400 micrograms paraffin oil-silicon oil/min/5 X 10(6) PMN, which is about 20 times higher than that reported for the same cell type using paraffin oil particles. The calculated maximum phagocytic capacity was 2.5 mg paraffin oil-silicon oil/5 X 10(6) PMN. The uptake of paraffin oil-silicon oil particles was sensitive to inhibitors of phagocytosis, such as N-ethylmaleimide, papaverine and cytochalasin B, in a dose dependent manner. The assay also permitted the detection of increased phagocytosis such as occurs in myeloperoxidase deficient PMN.

Cell Separation↗
PubMed1985

Role of myeloperoxidase in respiratory burst of human polymorphonuclear leukocytes. Studies with myeloperoxidase-deficient subjects.

A comparative study of the respiratory burst [monitored as superoxide (O2-) production] of normal and myeloperoxidase (MPO) -deficient polymorphonuclear leukocytes (PMNs) was carried out on 11 MPO-deficient subjects that represent the largest sample of this kind ever studied. The rate of O2- production by isolated PMNs and whole blood from normal and MPO-deficient subjects was comparable during the initial 30-40 min of incubation with serum-treated zymosan (STZ). Afterwards, the amount of O2- produced became progressively higher in MPO-deficient cells at least until 120 min incubation with STZ. On the contrary the rate of O2- production by both cell types in response to 4-beta-phorbol-12-myristate-13-acetate (PMA) was the same. The PMNs of four MPO-deficient subjects were tested for their ingestion ability by counting the number of ingested particles on toluidine blue-stained sections of epoxy-embedded PMN suspensions. Both cell types ingested STZ particles at a comparable rate at early postphagocytic times, whereas on prolonged incubation MPO-deficient PMNs ingested more STZ particles than normal PMNs. These results suggest that the ingestion capacity of normal cells may undergo a more rapid deterioration than that of MPO-deficient cells during incubation with STZ. Evidence for a higher deterioration of normal PMNs with respect to MPO-deficient PMNs was obtained also from studies on the effect of storage on O2- generation. After standing at melting ice temperature for 3 h, normal PMNs produced less O2- than MPO-deficient PMNs in response to PMA, and the difference in O2- production by the two cell types in response to STZ was evident at earlier postphagocytic periods than with freshly isolated cells. Taken all together these results suggest that normal PMNs and MPO-deficient PMNs do not intrinsically differ in O2- generating potential and that the difference in the respiratory burst observed during phagocytosis may be accounted for by a more marked deterioration, in normal PMNs, of one or more functions related to the respiratory burst.

Humans↗
PubMed1985

Increased degranulation of human myeloperoxidase-deficient polymorphonuclear leucocytes.

Myeloperoxidase (MPO)-deficient neutrophils (PMN) released considerably more beta-glucuronidase, lysozyme and vitamin B12-binding activities, when exposed to opsonized zymosan (STZ), than the normal counterpart. Release of the soluble enzyme lactate dehydrogenase was not appreciably changed over the incubation time with particles in either cell type. MPO-deficient PMN and normal PMN ingested STZ particles at a similar rate at early times, but thereafter phagocytosis by MPO-deficient PMN was significantly higher than that by normal PMN. The difference in degranulation between the two cell types greatly exceeded the difference in ingestion and was evident already at early phagocytosis times when no difference in phagocytosis was observed; this suggested that the higher degranulation in MPO-deficient PMN was at least in part independent of the increased ingestion. This was confirmed by experiments with the soluble stimulant N-formyl-L-norleucyl-L-leucyl-phenylalanine (FNLLP). MPO-deficient PMN and normal PMN exhibited a comparable respiratory burst when exposed to FNLLP plus cytochalasin B, but the defective cells released more azurophilic and specific granule markers than normal PMN. These results indicate that MPO-deficient PMN degranulate more than normal PMN and suggest a role for MPO in the regulation of degranulation.

Cytoplasmic Granules↗